Dataset Information

Real-time observation of strand exchange reaction with high spatiotemporal resolution.

ABSTRACT: RecA binds to single-stranded (ss) DNA to form a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggest that propagation occurs in 3 bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Unexpectedly, we discovered the formation of a dynamic complex between RecA and the displaced DNA that remains bound transiently after joint molecule formation. This finding could have important implications for the irreversibility of strand exchange. Our model for strand exchange links structural models of RecA to its catalytic function.

SUBMITTER: Ragunathan K

PROVIDER: S-EPMC3163668 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Real-time observation of strand exchange reaction with high spatiotemporal resolution.

Ragunathan Kaushik K Joo Chirlmin C Ha Taekjip T

Structure (London, England : 1993) 20110801 8

RecA binds to single-stranded (ss) DNA to form a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our ...[more]

PMID: 21827943

Similar Datasets

Project description:Single-cell metabolic investigations are hampered by the absence of flexible tools to measure local partial pressure of O2 (pO2) at high spatial-temporal resolution. To this end, we developed an optical sensor capable of measuring local pericellular pO2 for subcellular resolution measurements with confocal imaging while simultaneously carrying out electrophysiological and/or chemo-mechanical single cell experiments. Here we present the OxySplot optrode, a ratiometric fluorescent O2-micro-sensor created by adsorbing O2-sensitive and O2-insensitive fluorophores onto micro-particles of silica. To protect the OxySplot optrode from the components and reactants of liquid environment without compromising access to O2, the micro-particles are coated with an optically clear silicone polymer (PDMS, polydimethylsiloxane). The PDMS coated OxySplot micro-particles are used alone or in a thin (~50 μm) PDMS layer of arbitrary shape referred to as the OxyMat. Additional top coatings on the OxyMat (e.g., fibronectin, laminin, polylysine, special photoactivatable surfaces etc.) facilitate adherence of cells. The OxySplots report the cellular pO2 and micro-gradients of pO2 without disrupting the flow of extracellular solutions or interfering with patch-clamp pipettes, mechanical attachments, and micro-superfusion. Since OxySplots and a cell can be imaged and spatially resolved, calibrated changes of pO2 and intracellular events can be imaged simultaneously. In addition, the response-time (t0.5 = 0.7 s, 0-160 mmHg) of OxySplots is ~100 times faster than amperometric Clark-type polarization microelectrodes. Two usage example of OxySplots with cardiomyocytes show (1) OxySplots measuring pericellular pO2 while tetramethylrhodamine methyl-ester (TMRM) was used to measure mitochondrial membrane potential (ΔΨm); and (2) OxySplots measuring pO2 during ischemia and reperfusion while rhod-2 was used to measure cytosolic [Ca2+]i levels simultaneously. The OxySplot/OxyMat optrode system provides an affordable and highly adaptable optical sensor system for monitoring pO2 with a diverse array of imaging systems, including high-speed, high-resolution confocal microscopes while physiological features are measured simultaneously.

Project description:BackgroundReal-time cardiovascular magnetic resonance (CMR) assessment of ventricular volumes and function enables data acquisition during free-breathing. The requirement for high spatiotemporal resolution in children necessitates the use of highly accelerated imaging techniques.MethodsA novel real-time balanced steady state free precession (bSSFP) spiral sequence reconstructed using Compressed Sensing (CS) was prospectively validated against the breath-hold clinical standard for assessment of ventricular volumes in 60 children with congenital heart disease. Qualitative image scoring, quantitative image quality, as well as evaluation of biventricular volumes was performed. Standard BH and real-time measures were compared using the paired t-test and agreement for volumetric measures were evaluated using Bland Altman analysis.ResultsAcquisition time for the entire short axis stack (~?13 slices) using the spiral real-time technique was ~?20 s, compared to ~?348 s for the standard breath hold technique. Qualitative scores reflected more residual aliasing artefact (p?<?0.001) and lower edge definition (p?<?0.001) in spiral real-time images than standard breath hold images, with lower quantitative edge sharpness and estimates of image contrast (p?<?0.001). There was a small but statistically significant (p?<?0.05) overestimation of left ventricular (LV) end-systolic volume (1.0?±?3.5 mL), and underestimation of LV end-diastolic volume (-?1.7?±?4.6 mL), LV stroke volume (-?2.6?±?4.8 mL) and LV ejection fraction (-?1.5?±?3.0%) using the real-time technique. We also observed a small underestimation of right ventricular stroke volume (-?1.8?±?4.9 mL) and ejection fraction (-?1.4?±?3.7%) using the real-time imaging technique. No difference in inter-observer or intra-observer variability were observed between the BH and real-time sequences.ConclusionsReal-time bSSFP imaging using spiral trajectories combined with a compressed sensing reconstruction showed good agreement for quantification of biventricular metrics in children with heart disease, despite slightly lower image quality. This technique holds the potential for free breathing data acquisition, with significantly shorter scan times in children.

Dataset Information

Real-time observation of strand exchange reaction with high spatiotemporal resolution.

Publications

Real-time observation of strand exchange reaction with high spatiotemporal resolution.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets